Methods of detecting a liver disorder

ABSTRACT

Growth differentiation factor-12 (GDF-12) is disclosed along with its polynucleotide sequence and amino acid sequence. Also disclosed are diagnostic and therapeutic methods of using the GDF-12 polypeptide and polynucleotide sequences.

[0001] This is a continuation-in-part application of U.S. Ser. No.08/274,215, filed on Jul. 13, 1994.

BACKGROUND OF THE INVENTION

[0002] 1. Field of the Invention

[0003] The invention relates generally to growth factors andspecifically to a new member of the transforming growth factor beta(TGF-β) superfamily, which is denoted, growth differentiation factor-12(GDF-12).

[0004] 2. Description of Related Art

[0005] The transforming growth factor β (TGF-β) superfamily encompassesa group of structurally-related proteins which affect a wide range ofdifferentiation processes during embryonic development. The familyincludes, Mullerian inhibiting substance (MIS), which is required fornormal male sex development (Behringer, et al., Nature, 345:167, 1990),Drosophila decapentaplegic (DPP) gene product, which is required fordorsal-ventral axis formation and morphogenesis of the imaginal disks(Padgett, et al., Nature, 325:81-84, 1987), the Xenopus Vg-1 geneproduct, which localizes to the vegetal pole of eggs (Weeks, et al.,Cell, 51:861-867, 1987), the activins (Mason, et al., Biochem, Biophys.Res. Commun., 135:957-964,1986), which can induce the formation ofmesoderm and anterior structures in Xenopus embryos (Thomsen, et al.,Cell, 63:485, 1990), and the bone morphogenetic proteins (BMPS,osteogenin, OP-1) which can induce de novo cartilage and bone formation(Sampath, et al., J. Biol. Chem., 265:13198, 1990). The TGF-βs caninfluence a variety of differentiation processes, includingadipogenesis, myogenesis, chondrogenesis, hematopoiesis, and epithelialcell differentiation (for review, see Massague, Cell 49:437, 1987).

[0006] The proteins of the TGF-β family are initially synthesized as alarge precursor protein which subsequently undergoes proteolyticcleavage at a cluster of basic residues approximately 110-140 aminoacids from the C-terminus. The C-terminal regions, or mature regions, ofthe proteins are all structurally related and the different familymembers can be classified into distinct subgroups based on the extent oftheir homology. Although the homologies within particular subgroupsrange from 70% to 90% amino acid sequence identity, the homologiesbetween subgroups are significantly lower, generally ranging from only20% to 50%. In each case, the active species appears to be adisulfide-linked dimer of C-terminal fragments. Studies have shown thatwhen the pro-region of a member of the TGF-β family is coexpressed witha mature region of another member of the TGF-β family, intracellulardimerization and secretion of biologically active homodimers occur(Gray, A., and Maston, A, Science, 247:1328,1990). Additional studies byHammonds, et a., (Molec. Endocrin. 5:149, 1991) showed that the use ofthe BMP-2 pro-region combined with the BMP-4 mature region led todramatically improved expression of mature BMP-4. For most of the familymembers that have been studied, the homodimeric species has been foundto be biologically active, but for other family members, like theinhibins (Ling, et al., Nature, 321:779, 1986) and the TGF-βs (Cheifetz,et al., Cell, 48:409, 1987), heterodimers have also been detected, andthese appear to have different biological properties than the respectivehomodimers.

[0007] Identification of new factors that are tissue-specific in theirexpression pattern will provide a greater understanding of that tissue'sdevelopment and function.

SUMMARY OF THE INVENTION

[0008] The present invention provides a cell growth and differentiationfactor, GDF-12, a polynucleotide sequence which encodes the factor, andantibodies which are immunoreactive with the factor. This factor appearsto relate to various cell proliferative disorders, especially thoseinvolving liver cells.

[0009] Thus, in one embodiment, the invention provides a method fordetecting a cell proliferative disorder of liver origin and which isassociated with GDF-12. In another embodiment, the invention provides amethod for treating a cell proliferative disorder by suppressing orenhancing GDF-12 activity.

BRIEF DESCRIPTION OF THE DRAWINGS

[0010]FIG. 1 shows a Northern blot of RNA prepared from adult tissuesprobed with a murine GDF-12 probe.

[0011]FIG. 2 shows the partial nucleotide and predicted amino acidsequence of human GDF-12.

[0012]FIG. 3 shows the full length nucleotide and predicted amino acidsequence of human GDF-12.

[0013]FIG. 4 shows amino acid sequence homologies between human GDF-12and different members of the TGF-β superfamily. Numbers represent aminoacid sequence identities between GDF-12 and the indicated family membercalculated from the first conserved cysteine to the C-terminus.

DETAILED DESCRIPTION OF THE INVENTION

[0014] The present invention provides a growth and differentiationfactor, GDF-12 and a polynucleotide sequence encoding GDF-12. GDF-12 isexpressed specifically in liver. In one embodiment, the inventionprovides a method for detection of a cell proliferative disorder ofliver origin which is associated with GDF-12 expression. In anotherembodiment, the invention provides a method for treating a cellproliferative disorder by using an agent which suppresses or enhancesGDF-12 activity.

[0015] The TGF-β superfamily consists of multifunctional polypeptidesthat control proliferation, differentiation, and other functions in manycell types. Many of the peptides have regulatory, both positive andnegative, effects on other peptide growth factors. The structuralhomology between the GDF-12 protein of this invention and the members ofthe TGF-β family, indicates that GDF-12 is a new member of the family ofgrowth and differentiation factors. Based on the known activities ofmany of the other members, it can be expected that GDF-12 will alsopossess biological activities that will make it useful as a diagnosticand therapeutic reagent.

[0016] In particular, the expression pattern of GDF-12 suggests thatGDF-12 possesses activities that will make it useful for the treatmentof various diseases involving the liver. For example, when GDF-12functions to stimulate the growth or differentiation of liver cells,GDF-12 could be used for the treatment of disease states in which thefunction of the liver is compromised, such as in hepatitis or cirrhosis.Although liver tissue has the capacity to regenerate, GDF-12 couldpotentially accelerate the normal regenerative process or promote theprocess in disease states in which the regenerative process issuppressed. Similarly, GDF-12 could be useful for maintaining livercells or tissue in culture prior to transplantation or for stimulatingthe growth of liver cells following transplantation; in this regard,because liver cells may be used as a vehicle for delivering genes toliver for gene therapy, GDF-12 could be useful for maintaining orexpanding liver cells in culture during or after the introduction ofparticular genes or for stimulating the growth of these cells followingtransplantation.

[0017] Alternatively, when GDF-12 functions as a growth inhibitor,GDF-12 could be used to create cell proliferative disorders involvingliver cells, such as hepatocellular carcinoma. Indeed, one member ofthis superfamily, namely, inhibin alpha, has been shown to function as atumor suppressor gene, and another member of this superfamily, namely,Mullerian inhibiting substance, has been shown to be capable ofinhibiting the growth of tumor cells both in vitro and in vivo.

[0018] This high specificity of GDF-12 expression also suggestspotential applications of GDF-12 as a diagnostic tool. In particular,because GDF-12 encodes a secreted factor, levels of GDF-12 could be usedto monitor liver function or to detect the presence of neoplasmsinvolving liver cells. In this regard, another member of this family,namely, inhibin, has been shown to be useful as a marker for ovariangranulosa cell tumors.

[0019] The term “substantially pure” as used herein refers to GDF-12which is substantially free of other proteins, lipids, carbohydrates orother materials with which it is naturally associated. One skilled inthe art can purify GDF-12 using standard techniques for proteinpurification. The substantially pure polypeptide will yield a singlemajor band on a non-reducing polyacrylamide gel. The purity of theGDF-12 polypeptide can also be determined by amino-terminal amino acidsequence analysis. GDF-12 polypeptide includes functional fragments ofthe polypeptide, as long as the activity of GDF-12 remains. Smallerpeptides containing the biological activity of GDF-12 are included inthe invention.

[0020] The invention provides polynucleotides encoding the GDF-12protein. These polynucleotides include DNA, cDNA and RNA sequences whichencode GDF-12. It is understood that all polynucleotides encoding all ora portion of GDF-12 are also included herein, as long as they encode apolypeptide with GDF-12 activity. Such polynucleotides include naturallyoccurring, synthetic, and intentionally manipulated polynucleotides. Forexample, GDF-12 polynucleotide may be subjected to site-directedmutagenesis. The polynucleotide sequence for GDF-12 also includesantisense sequences. The polynucleotides of the invention includesequences that are degenerate as a result of the genetic code. There are20 natural amino acids, most of which are specified by more than onecodon. Therefore, all degenerate nucleotide sequences are included inthe invention as long as the amino acid sequence of GDF-12 polypeptideencoded by the nucleotide sequence is functionally unchanged.

[0021] Specifically disclosed herein is a partial cDNA sequencecontaining the active portion of the human GDF-12 coding sequence. Oneof skill in the art could now use this partial sequence to isolate thefull length clones. The cDNA clone from which this sequence was obtainedis likely to contain the entire coding sequence for GDF-12 . Thedisclosed sequence corresponds to the C-terminal region of the GDF-12polypeptide. The sequence begins with a putative proteolytic cleavagesite, RARRR. Cleavage of the polypeptide at this site would generate anactive C-terminal fragment 114 amino acids in length with a predictedmolecular weight of 12,500.

[0022] The C-terminal region of GDF-12 following the putativeproteolytic processing site shows significant homology to the knownmembers of the TGF-β superfamily. The GDF-12 sequence contains most ofthe residues that are highly conserved in other family members (see FIG.1). Like the TGF-βs and inhibin βs, GDF-12 contains an extra pair ofcysteine residues in addition to the 7 cysteines found in virtually allother family members. Among the known family members, GDF-12 is mosthomologous to Inhibin βB (50% sequence identity) (see FIG. 4).

[0023] Minor modifications of the recombinant GDF-12 primary amino acidsequence may result in proteins which have substantially equivalentactivity as compared to the GDF-12 polypeptide described herein, Suchmodifications may be deliberate, as by site-directed mutagenesis, or maybe spontaneous. All of the polypeptides produced by these modificationsare included herein as long as the biological activity of GDF-12 stillexists. Further, deletion of one or more amino acids can also result ina modification of the structure of the resultant molecule withoutsignificantly altering its biological activity. This can lead to thedevelopment of a smaller active molecule which would have broaderutility. For example, one can remove amino or carboxy terminal aminoacids which are not required for GDF-12 biological activity.

[0024] The nucleotide sequence encoding the GDF-12 polypeptide of theinvention includes the disclosed sequence and conservative variationsthereof. The term “conservative variation” as used herein denotes thereplacement of an amino acid residue by another, biologically similarresidue. Examples of conservative variations include the substitution ofone hydrophobic residue such as isoleucine, valine, ieucine ormethionine for another, or the substitution of one polar residue foranother, such as the substitution of arginine for iysine, glutamic foraspartic acid, or glutamine for asparagine, and the like. The term“conservative variation” also includes the use of a substituted aminoacid in place of an unsubstituted parent amino acid provided thatantibodies raised to the substituted polypeptide also immunoreact withthe unsubstituted polypeptide.

[0025] The polynucleotide encoding GDF-12 includes the nucleotidesequence in FIGS. 2 and 3 (SEQ ID NO:11 and NO:13, respectively), aswell as nucleic acid sequences complementary to that sequence. Acomplementary sequence may include an antisense nucleotide. When thesequence is RNA, the deoxynucleotides A, G, C, and T of FIGS. 2 and 3are replaced by ribonucleotides A, G, C, and U, respectively. Alsoincluded in the invention are fragments of the above-described nucleicacid sequences that are at least 15 bases in length, which is sufficientto permit the fragment to selectively hybridize to DNA that encodes theprotein of FIGS. 2 and 3 (SEQ ID NO:12 and NO:14, respectively) underphysiological conditions.

[0026] DNA sequences of the invention can be obtained by severalmethods. For example, the DNA can be isolated using hybridizationtechniques which are well known in the art. These include, but are notlimited to: 1) hybridization of genomic or cDNA libraries with probes todetect homologous nucleotide sequences, 2) polymerase chain reaction(PCR) on genomic DNA or cDNA using primers capable of annealing to theDNA sequence of interest, and 3) antibody screening of expressionlibraries to detect cloned DNA fragments with shared structuralfeatures.

[0027] Preferably the GDF-12 polynucleotide of the invention is derivedfrom a mammalian organism, and most preferably from a mouse, rat, orhuman. Screening procedures which rely on nucleic acid hybridizationmake it possible to isolate any gene sequence from any organism,provided the appropriate probe is available. Oligonucleotide probes,which correspond to a part of the sequence encoding the protein inquestion, can be synthesized chemically. This requires that short,oligopeptide stretches of amino acid sequence must be known. The DNAsequence encoding the protein can be deduced from the genetic code,however, the degeneracy of the code must be taken into account. It ispossible to perform a mixed addition reaction when the sequence isdegenerate. This includes a heterogeneous mixture of denatureddouble-stranded DNA. For such screening, hybridization is preferablyperformed on either single-stranded DNA or denatured double-strandedDNA. Hybridization is particularly useful in the detection of cDNAclones derived from sources where an extremely low amount of mRNAsequences relating to the polypeptide of interest are present. In otherwords, by using stringent hybridization conditions directed to avoidnon-specific binding, it is possible, for example, to allow theautoradiographic visualization of a specific cDNA clone by thehybridization of the target DNA to that single probe in the mixturewhich is its complete complement (Wallace, et al., Nucl. Acid Res.,9:879, 1981).

[0028] Therefore, given a partial DNA sequence of the gene of interest,one of skill in the art would be able to prepare probes for isolation ofa full length cDNA clone, without undue experimentation (see forexample, Ausubel, et al., Current Protocols in Molecular Biology, Units6.3-6.4, Greene Pubi., 1994; Maniatis, et al., Molecular Cloning, ColdSpring Harbor Laboratories, 1982).

[0029] The development of specific DNA sequences encoding GDF-12 canalso be obtained by: 1) isolation of double-stranded DNA sequences fromthe genomic DNA; 2) chemical manufacture of a DNA sequence to providethe necessary codons for the polypeptide of interest; and 3) in vitrosynthesis of a double-stranded DNA sequence by reverse transcription ofmRNA isolated from a eukaryotic donor cell. In the latter case, adouble-stranded DNA complement of mRNA is eventually formed which isgenerally referred to as cDNA.

[0030] Of the three above-noted methods for developing specific DNAsequences for use in recombinant procedures, the isolation of genomicDNA isolates is the least common. This is especially true when it isdesirable to obtain the microbial expression of mammalian polypeptidesdue to the presence of introns.

[0031] The synthesis of DNA sequences is frequently the method of choicewhen the entire sequence of amino acid residues of the desiredpolypeptide product is known. When the entire sequence of amino acidresidues of the desired polypeptide is not known, the direct synthesisof DNA sequences is not possible and the method of choice is thesynthesis of cDNA sequences. Among the standard procedures for isolatingcDNA sequences of interest is the formation of plasmid—or phage-carryingcDNA libraries which are derived from reverse transcription of mRNAwhich is abundant in donor cells that have a high level of geneticexpression. When used in combination with polymerase chain reactiontechnology, even rare expression products can be cloned. In those caseswhere significant portions of the amino acid sequence of the polypeptideare known, the production of labeled single or double-stranded DNA orRNA probe sequences duplicating a sequence putatively present in thetarget cDNA may be employed in DNA/DNA hybridization procedures whichare carried out on cloned copies of the cDNA which have been denaturedinto a single-stranded form (Jay, et al., Nucl. Acid Res., 11:2325,1983).

[0032] A cDNA expression library, such as lambda gt11, can be screenedindirectly for GDF-12 peptides having at least one epitope, usingantibodies specific for GDF-12. Such antibodies can be eitherpolyclonally or monocionally derived and used to detect expressionproduct indicative of the presence of GDF-12 cDNA.

[0033] DNA sequences encoding GDF-12 can be expressed in vitro by DNAtransfer into a suitable host cell. “Host cells” are cells in which avector can be propagated and its DNA expressed. The term also includesany progeny of the subject host cell. It is understood that all progenymay not be identical to the parental cell since there may be mutationsthat occur during replication. However, such progeny are included whenthe term “host cell” is used. Methods of stable transfer, meaning thatthe foreign DNA is continuously maintained in the host, are known in theart.

[0034] In the present invention, the GDF-12 polynucleotide sequences maybe inserted into a recombinant expression vector. The term “recombinantexpression vector” refers to a plasmid, virus or other vehicle known inthe art that has been manipulated by insertion or incorporation of theGDF-12 genetic sequences. Such expression vectors contain a promotersequence which facilitates the efficient transcription of the insertedgenetic sequence of the host. The expression vector typically containsan origin of replication, a promoter, as well as specific genes whichallow phenotypic selection of the transformed cells. Vectors suitablefor use in the present invention include, but are not limited to theT7-based expression vector for expression in bacteria (Rosenberg, etal., Gene, 56:125, 1987), the pMSXND expression vector for expression inmammalian cells (Lee and Nathans, J. Biol. Chem., 263:3521, 1988) andbaculovirus-derived vectors for expression in insect cells. The DNAsegment can be present in the vector operably linked to regulatoryelements, for example, a promoter (e.g., T7, metallothionein I, orpolyhedrin promoters).

[0035] Polynucleotide sequences encoding GDF-12 can be expressed ineither prokaryotes or eukaryotes. Hosts can include microbial, yeast,insect and mammalian organisms. Methods of expressing DNA sequenceshaving eukaryotic or viral sequences in prokaryotes are well known inthe art. Biologically functional viral and plasmid DNA vectors capableof expression and replication in a host are known in the art. Suchvectors are used to incorporate DNA sequences of the invention.Preferably, the mature C-terminal region of GDF-12 is expressed from acDNA clone containing entire coding sequence of GDF-12. Alternatively,the C-terminal portion of GDF-12 can be expressed as a fusion proteinwith the pro-region of another member of the TGF-β family orco-expressed with another pro-region (see for example, Hammonds, et al.,Molec. Endocrin. 5:149, 1991; Gray, A., and Mason, A., Science,247:1328, 1990).

[0036] Transformation of a host cell with recombinant DNA may be carriedout by conventional techniques as are well known to those skilled in theart. Where the host is prokaryotic, such as E. coli, competent cellswhich are capable of DNA uptake can be prepared from cells harvestedafter exponential growth phase and subsequently treated by the CaCl₂method using procedures well known in the art. Alternatively, MgCl₂ orRbCl can be used. Transformation can also be performed after forming aprotoplast of the host cell if desired.

[0037] When the host is a eukaryote, such methods of transfection of DNAas calcium phosphate co-precipitates, conventional mechanical proceduressuch as microinjection, electroporation, insertion of a plasmid encasedin liposomes, or virus vectors may be used. Eukaryotic cells can also becotransformed with DNA sequences encoding the GDF-12 of the invention,and a second foreign DNA molecule encoding a selectable phenotype, suchas the herpes simplex thymidine kinase gene. Another method is to use aeukaryotic viral vector, such as simian virus 40 (SV40) or bovinepapilloma virus, to transiently infect or transform eukaryotic cells andexpress the protein. (see for example, Eukaryotic Viral Vectors, ColdSpring Harbor Laboratory, Gluzman ed., 1982).

[0038] Isolation and purification of microbial expressed polypeptide, orfragments thereof, provided by the invention, may be carried out byconventional means including preparative chromatography andimmunological separations involving monoclonal or polyclonal antibodies.

[0039] The GDF-12 polypeptides of the invention can also be used toproduce antibodies which are immunoreactive or bind to epitopes of theGDF-12 polypeptides. Antibody which consists essentially of pooledmonoclonal antibodies with different epitopic specificities, as well asdistinct monoclonal antibody preparations are provided. Monoclonalantibodies are made from antigen containing fragments of the protein bymethods well known in the art (Kohler, et al, Nature, 256:495, 1975;Current Protocols in Molecular Biology, Ausubel, et al., ed., 1989).

[0040] The term “antibody” as used in this invention includes intactmolecules as well as fragments thereof, such as Fab, F(ab′)₂, and Fvwhich are capable of binding the epitopic determinant. These antibodyfragments retain some ability to selectively bind with its antigen orreceptor and are defined as follows:

[0041] (1) Fab, the fragment which contains a monovalent antigen-bindingfragment of an antibody molecule can be produced by digestion of wholeantibody with the enzyme papain to yield an intact light chain and aportion of one heavy chain;

[0042] (2) Fab′, the fragment of an antibody molecule can be obtained bytreating whole antibody with pepsin, followed by reduction, to yield anintact light chain and a portion of the heavy chain; two Fab′ fragmentsare obtained per antibody molecule;

[0043] (3) (Fab′)₂, the fragment of the antibody that can be obtained bytreating whole antibody with the enzyme pepsin without subsequentreduction; F(ab′)₂ is a dimer of two Fab′ fragments held together by twodisulfide bonds;

[0044] (4) Fv, defined as a genetically engineered fragment containingthe variable region of the light chain and the variable region of theheavy chain expressed as two chains; and

[0045] (5) Single chain antibody (“SCA”), defined as a geneticallyengineered molecule containing the variable region of the light chain,the variable region of the heavy chain, linked by a suitable polypeptidelinker as a genetically fused single chain molecule.

[0046] Methods of making these fragments are known in the art. (See forexample, Harlow and Lane, Antibodies: A Laboratory Manual, Cold SpringHarbor Laboratory, N.Y. (1988), incorporated herein by reference).

[0047] As used in this invention, the term “epitope” means any antigenicdeterminant on an antigen to which the paratope of an antibody binds.Epitopic determinants usually consist of chemically active surfacegroupings of molecules such as amino acids or sugar side chains andusually have specific three dimensional structural characteristics, aswell as specific charge characteristics.

[0048] Antibodies which bind to the GDF-12 polypeptide of the inventioncan be prepared using an intact polypeptide or fragments containingsmall peptides of interest as the immunizing antigen. The polypeptide ora peptide used to immunize an animal can be derived from translated cDNAor chemical synthesis which can be conjugated to a carrier protein, ifdesired. Such commonly used carriers which are chemically coupled to thepeptide include keyhole limpet hemocyanin (KLH), thyroglobulin, bovineserum albumin (BSA), and tetanus toxoid. The coupled peptide is thenused to immunize the animal (e.g., a mouse, a rat, or a rabbit).

[0049] If desired, polyclonal or monoclonal antibodies can be furtherpurified, for example, by binding to and elution from a matrix to whichthe polypeptide or a peptide to which the antibodies were raised isbound. Those of skill in the art will know of various techniques commonin the immunology arts for purification and/or concentration ofpolyclonal antibodies, as well as monoclonal antibodies (See forexample, Coligan, et al., Unit 9, Current Protocols in Immunology, Wileyinterscience, 1991, incorporated by reference).

[0050] It is also possible to use the anti-idiotype technology toproduce monoclonal antibodies which mimic an epitope. For example, ananti-idiotypic monoclonal antibody made to a first monoclonal antibodywill have a binding domain in the hypervariable region which is the“image” of the epitope bound by the first monoclonal antibody.

[0051] The term “cell-proliferative disorder” denotes malignant as wellas non-malignant cell populations which often appear to differ from thesurrounding tissue both morphologically and genotypically. Malignantcells (i.e. cancer) develop as a result of a multistep process. TheGDF-12 polynucleotide that is an antisense molecule is useful intreating malignancies of the various organ systems, particularly, forexample, cells in liver tissue. Essentially, any disorder which isetiologically linked to altered expression of GDF-12 could be consideredsusceptible to treatment with a GDF-12 suppressing reagent. One suchdisorder is a malignant cell proliferative disorder, for example.

[0052] The invention provides a method for detecting a cellproliferative disorder of muscle or adipose tissue which comprisescontacting an anti-GDF-12 antibody with a cell suspected of having aGDF-12 associated disorder and detecting binding to the antibody. Theantibody reactive with GDF-12 is labeled with a compound which allowsdetection of binding to GDF-12. For purposes of the invention, anantibody specific for GDF-12 polypeptide may be used to detect the levelof GDF-12 in biological fluids and tissues. Any specimen containing adetectable amount of antigen can be used. A preferred sample of thisinvention is liver tissue. The level of GDF-12 in the suspect cell canbe compared with the level in a normal cell to determine whether thesubject has a GDF-12-associated cell proliferative disorder. Preferablythe subject is human.

[0053] The antibodies of the invention can be used in any subject inwhich it is desirable to administer in vitro or in vivo immunodiagnosisor immunotherapy. The antibodies of the invention are suited for use,for example, in immunoassays in which they can be utilized in liquidphase or bound to a solid phase carrier. In addition, the antibodies inthese immunoassays can be detectably labeled in various ways. Examplesof types of immunoassays which can utilize antibodies of the inventionare competitive and non-competitive immunoassays in either a direct orindirect format. Examples of such immunoassays are the radioimmunoassay(RIA) and the sandwich (immunometric) assay. Detection of the antigensusing the antibodies of the invention can be done utilizing immunoassayswhich are run in either the forward, reverse, or simultaneous modes,including immunohistochemical assays on physiological samples. Those ofskill in the art will know, or can readily discern, other immunoassayformats without undue experimentation.

[0054] The antibodies of the invention can be bound to many differentcarriers and used to detect the presence of an antigen comprising thepolypeptide of the invention. Examples of well-known carriers includeglass, polystyrene, polypropylene, polyethylene, dextran, nylon,amylases, natural and modified celluloses, polyacrylamides, agaroses andmagnetite. The nature of the carrier can be either soluble or insolublefor purposes of the invention. Those skilled in the art will know ofother suitable carriers for binding antibodies, or will be able toascertain such, using routine experimentation.

[0055] There are many different labels and methods of labeling known tothose of ordinary skill in the art. Examples of the types of labelswhich can be used in the present invention include enzymes,radioisotopes, fluorescent compounds, colloidal metals, chemiluminescentcompounds, phosphorescent compounds, and bioluminescent compounds. Thoseof ordinary skill in the art will know of other suitable labels forbinding to the antibody, or will be able to ascertain such, usingroutine experimentation.

[0056] Another technique which may also result in greater sensitivityconsists of coupling the antibodies to low molecular weight haptens.These haptens can then be specifically detected by means of a secondreaction. For example, it is common to use such haptens as biotin, whichreacts with avidin, or dinitrophenyt, puridoxal, and fluorescein, whichcan react with specific anti-hapten antibodies.

[0057] In using the monoclonal antibodies of the invention for the invivo detection of antigen, the detectably labeled antibody is given adose which is diagnostically effective. The term “diagnosticallyeffective” means that the amount of detectably labeled monoclonalantibody is administered in sufficient quantity to enable detection ofthe site having the antigen comprising a polypeptide of the inventionfor which the monoclonal antibodies are specific.

[0058] The concentration of detectably labeled monoclonal antibody whichis administered should be sufficient such that the binding to thosecells having the polypeptide is detectable compared to the background.Further, it is desirable that the detectably-labeled monoclonal antibodybe rapidly cleared from the circulatory system in order to give the besttarget-to-background signal ratio.

[0059] As a rule, the dosage of detectably labeled monoclonal antibodyfor in vivo diagnosis will vary depending on such factors as age, sex,and extent of disease of the individual. Such dosages may vary, forexample, depending on whether multiple injections are given, antigenicburden, and other factors known to those of skill in the art.

[0060] For in vivo diagnostic imaging, the type of detection instrumentavailable is a major factor in selecting a given radioisotope. Theradioisotope chosen must have a type of decay which is detectable for agiven type of instrument. Still another important factor in selecting aradioisotope for in vivo diagnosis is that deleterious radiation withrespect to the host is minimized. Ideally, a radioisotope used for invivo imaging will lack a particle emission, but produce a large numberof photons in the 140-250 keV range, which may readily be detected byconventional gamma cameras.

[0061] For in vivo diagnosis radioisotopes may be bound toimmunoglobulin either directly or indirectly by using an intermediatefunctional group. Intermediate functional groups which often are used tobind radioisotopes which exist as metallic ions to immunoglobulins arethe bifunctional chelating agents such as diethylenetriaminepentaceticacid (DTPA) and ethylenediaminetetraacetic acid (EDTA) and similarmolecules. Typical examples of metallic ions which can be bound to themonoclonal antibodies of the invention are ¹¹¹In, ⁹⁷Ru, ⁶⁷Ga, ⁶⁸Ga,⁷²As, ⁸⁹Zr, and ²⁰¹Tl.

[0062] The monoclonal antibodies of the invention can also be labeledwith a paramagnetic isotope for purposes of in vivo diagnosis, as inmagnetic resonance imaging (MRI) or electron spin resonance (ESR). Ingeneral, any conventional method for visualizing diagnostic imaging canbe utilized. Usually gamma and positron emitting radioisotopes are usedfor camera imaging and paramagnetic isotopes for MRI. Elements which areparticularly useful in such techniques include ¹⁵⁷Gd, ⁵⁵Mn, ¹⁶²Dy, ⁵²Cr,and ⁵⁶Fe.

[0063] The monoclonal antibodies of the invention can be used in vitroand in vivo to monitor the course of amelioration of a GDF-12-associateddisease in a subject. Thus, for example, by measuring the increase ordecrease in the number of cells expressing antigen comprising apolypeptide of the invention or changes in the concentration of suchantigen present in various body fluids, it would be possible todetermine whether a particular therapeutic regimen aimed at amelioratingthe GDF-12-associated disease is effective. The term “ameliorate”denotes a lessening of the detrimental effect of the GDF-12-associateddisease in the subject receiving therapy.

[0064] The present invention identifies a nucleotide sequence that canbe expressed in an altered manner as compared to expression in a normalcell, therefore it is possible to design appropriate therapeutic ordiagnostic techniques directed to this sequence. Thus, where acell-proliferative disorder is associated with the expression of GDF-12, nucleic acid sequences that interfere with GDF-12 expression at thetranslational level can be used. This approach utilizes, for example,antisense nucleic acid and ribozymes to block translation of a specificGDF-12 mRNA, either by masking that mRNA with an antisense nucleic acidor by cleaving it with a ribozyme. Such disorders include liverdiseases, for example.

[0065] Antisense nucleic acids are DNA or RNA molecules that arecomplementary to at least a portion of a specific mRNA molecule(Weintraub, Scientific American, 262:40, 1990). In the cell, theantisense nucleic acids hybridize to the corresponding mRNA, forming adouble-stranded molecule. The antisense nucleic acids interfere with thetranslation of the mRNA, since the cell will not translate a mRNA thatis double-stranded. Antisense oligomers of about 15 nucleotides arepreferred, since they are easily synthesized and are less likely tocause problems than larger molecules when introduced into the targetGDF-12-producing cell. The use of antisense methods to inhibit the invitro translation of genes is well known in the art (Marcus-Sakura,Anal.Biochem., 172:289, 1988).

[0066] Ribozymes are RNA molecules possessing the ability tospecifically cleave other single-stranded RNA in a manner analogous toDNA restriction endonucleases. Through the modification of nucleotidesequences which encode these RNAs, it is possible to engineer moleculesthat recognize specific nucleotide sequences in an RNA molecule andcleave it (Cech, J.Amer.Med. Assn., 260:3030, 1988). A major advantageof this approach is that, because they are sequence-specific, only mRNAswith particular sequences are inactivated.

[0067] There are two basic types of ribozymes namely, tetrahymena-type(Hasselhoff, Nature, 334:585, 1988) and “hammerhead”-type.Tetrahymena-type ribozymes recognize sequences which are four bases inlength, while “hammerhead”-type ribozymes recognize base sequences 11-18bases in length. The longer the recognition sequence, the greater thelikelihood that the sequence will occur exclusively in the target mRNAspecies. Consequently, hammerhead-type ribozymes are preferable totetrahymena-type ribozymes for inactivating a specific mRNA species and18-based recognition sequences are preferable to shorter recognitionsequences.

[0068] The present invention also provides gene therapy for thetreatment of cell proliferative or immunologic disorders which aremediated by GDF-12 protein. Such therapy would achieve its therapeuticeffect by introduction of the GDF-12 antisense polynucleotide into cellshaving the proliferative disorder. Delivery of antisense GDF-12polynucleotide can be achieved using a recombinant expression vectorsuch as a chimeric virus or a colloidal dispersion system. Especiallypreferred for therapeutic delivery of antisense sequences is the use oftargeted liposomes.

[0069] Various viral vectors which can be utilized for gene therapy astaught herein include adenovirus, herpes virus, vaccinia, or,preferably, an RNA virus such as a retrovirus. Preferably, theretroviral vector is a derivative of a murine or avian retrovirus.Examples of retroviral vectors in which a single foreign gene can beinserted include, but are not limited to: Moloney murine leukemia virus(MoMuLV), Harvey murine sarcoma virus (HaMuSV), murine mammary tumorvirus (MuMTV), and Rous Sarcoma Virus (RSV). A number of additionalretroviral vectors can incorporate multiple genes. All of these vectorscan transfer or incorporate a gene for a selectable marker so thattransduced cells can be identified and generated. By inserting a GDF-12sequence of interest into the viral vector, along with another genewhich encodes the ligand for a receptor on a specific target cell, forexample, the vector is now target specific. Retroviral vectors can bemade target specific by inserting, for example, a polynucleotideencoding a sugar, a glycolipid, or a protein. Preferred targeting isaccomplished by using an antibody to target the retroviral vector. Thoseof skill in the art will know of, or can readily ascertain without undueexperimentation, specific polynucleotide sequences which can be insertedinto the retroviral genome to allow target specific delivery of theretroviral vector containing the GDF-12 antisense polynucleotide.

[0070] Since recombinant retroviruses are defective, they requireassistance in order to produce infectious vector particles. Thisassistance can be provided, for example, by using helper cell lines thatcontain plasmids encoding all of the structural genes of the retrovirusunder the control of regulatory sequences within the LTR. These plasmidsare missing a nucleotide sequence which enables the packaging mechanismto recognize an RNA transcript for encapsidation. Helper cell lineswhich have deletions of the packaging signal include, but are notlimited to ψ2, PA317 and PA12, for example. These cell lines produceempty virions, since no genome is packaged. If a retroviral vector isintroduced into such cells in which the packaging signal is intact, butthe structural genes are replaced by other genes of interest, the vectorcan be packaged and vector virion produced.

[0071] Alternatively, NIH 3T3 or other tissue culture cells can bedirectly transfected with plasmids encoding the retroviral structuralgenes gag, pol and env, by conventional calcium phosphate transfection.These cells are then transfected with the vector plasmid containing thegenes of interest. The resulting cells release the retroviral vectorinto the culture medium.

[0072] Another targeted delivery system for GDF-12 antisensepolynucleotides is a colloidal dispersion system. Colloidal dispersionsystems include macromolecule complexes, nanocapsules, microspheres,beads, and lipid-based systems including oil-in-water emulsions,micelles, mixed micelles, and liposomes. The preferred colloidal systemof this invention is a liposome. Liposomes are artificial membranevesicles which are useful as delivery vehicles in vitro and in vivo. Ithas been shown that large unilamellar vesicles (LUV), which range insize from 0.2-0.4 μm can encapsulate a substantial percentage of anaqueous buffer containing large macromolecules. RNA, DNA and intactvirions can be encapsulated within the aqueous interior and be deliveredto cells in a biologically active form (Fraley, et al., Trends Biochem.Sci., 6:77, 1981). In addition to mammalian cells, liposomes have beenused for delivery of polynucleotides in plant, yeast and bacterialcells. In order for a liposome to be an efficient gene transfer vehicle,the following characteristics should be present: (1) encapsulation ofthe genes of interest at high efficiency while not compromising theirbiological activity; (2) preferential and substantial binding to atarget cell in comparison to non-target cells; (3) delivery of theaqueous contents of the vesicle to the target cell cytoplasm at highefficiency; and (4) accurate and effective expression of geneticinformation (Mannino, et al., Biotechniques, 6:682, 1988).

[0073] The composition of the liposome is usually a combination ofphospholipids, particularly high-phase-transition-temperaturephospholipids, usually in combination with steroids, especiallycholesterol. Other phospholipids or other lipids may also be used. Thephysical characteristics of liposomes depend on pH, ionic strength, andthe presence of divalent cations.

[0074] Examples of lipids useful in liposome production includephosphatidyl compounds, such as phosphatidylglycerol,phosphatidylcholine, phosphatidylserine, phosphatidylethanolamine,sphingolipids, cerebrosides, and gangliosides. Particularly useful arediacylphosphatidylglycerols, where the lipid moiety contains from 14-18carbon atoms, particularly from 16-18 carbon atoms, and is saturated.Illustrative phospholipids include egg phosphatidylcholine,dipalmitoylphosphatidylcholine and distearoylphosphatidylcholine.

[0075] The targeting of liposomes can be classified based on anatomicaland mechanistic factors. Anatomical classification is based on the levelof selectivity, for example, organ-specific, cell-specific, andorganelle-specific. Mechanistic targeting can be distinguished basedupon whether it is passive or active. Passive targeting utilizes thenatural tendency of liposomes to distribute to cells of thereticulo-endothelial system (RES) in organs which contain sinusoidalcapillaries. Active targeting, on the other hand, involves alteration ofthe liposome by coupling the liposome to a specific ligand such as amonoclonal antibody, sugar, glycolipid, or protein, or by changing thecomposition or size of the liposome in order to achieve targeting toorgans and cell types other than the naturally occurring sites oflocalization.

[0076] The surface of the targeted delivery system may be modified in avariety of ways. In the case of a liposomal targeted delivery system,lipid groups can be incorporated into the lipid bilayer of the liposomein order to maintain the targeting ligand in stable association with theliposomal bilayer. Various linking groups can be used for joining thelipid chains to the targeting ligand.

[0077] Due to the expression of GDF-12 in liver tissue, there are avariety of applications using the polypeptide, polynucleotide, andantibodies of the invention, related to these tissues. Such applicationsinclude treatment of cell proliferative disorders involving this tissue.In addition, GDF-12 may be useful in various gene therapy procedures.

[0078] The following examples are intended to illustrate but not limitthe invention. While they are typical of those that might be used, otherprocedures known to those skilled in the art may alternatively be used.

EXAMPLE 1 Identification and Isolation of a Novel TGF-β Family Member

[0079] To identify novel members of the TGF-β superfamily, degenerateoligonucleotides were designed which Corresponded to two conservedregions among the known family members: one region spanning the twotryptophan residues conserved in most family members and the otherregion spanning the invariant cysteine residues near the C-terminus.These primers were used for polymerase chain reactions on cDNAsynthesized from RNA prepared from whole mouse embryos isolated at day18.5 of gestation. PCR products were subcloned using restriction sitesplaced at the 5′ ends of the primers, and individual bacterial coloniescarrying subcloned inserts were screened by a combination of randomsequencing and hybridization analysis to eliminate known members of thesuperfamily.

[0080] GDF-12 was identified from a mixture of PCR products obtainedwith combinations of primer: SJL218:5′-   CCGGAATTCGGITGG(C/A)G(G/A/T/C)(G/C/A)ATGG (SEQ ID NO:1)(A/G)TI(A/G)TITA(T/C)CC

[0081] with each of the following 9 primers: SJL188:5′-   CCGGAATTC(A/G)CAI(C/G)C(A/G)CAIC(C/T) (SEQ ID NO:2)(G/A/T/C)(T/A)CIACI(G/A)(T/C)CAT-3′ SJL190:5′-   CCGGAATTC(A/G)CAI(C/G)C(A/G)CAIT(C/G) (SEQ ID NO:3)(G/A/T/C)(C/T)GIACI(G/A)(T/C)CAT-3′ SJL191:5′-   CCGGAATTC(A/G)CAI(C/G)C(A/G)CAIT (SEQ ID NO:4)(C/G)(G/A/T/C)(T/A)CIACI(G/A)(T/C)CAT-3′ SJL192:5′-   CCGGAATTC(A/G)CAI(C/G)C(A/G)CAIT(C/G) (SEQ ID NO:5)(G/A/T/C)(C/G/T)TIACI(G/A)(T/C)CAT-3′ SJL193:5′-   CCGGAATTC(A/G)CAI(C/G)C(A/G)CAIG (SEQ ID NO:6)(A/C)(G/A/T/C)(C/T)GIACI(G/A)(T/C)CAT-3′ SJL194:5′-   CCGGAATTC(A/G)CAI(C/G)C(A/G)CAIG (SEQ ID NO:7)(A/C)(G/A/T/C)(T/A)CIACI(G/A)(T/C)CAT-3′ SJL196:5′-   CCGGAATTC(A/G)CAI(C/G)C(A/G)CAI(A/C)G (SEQ ID NO:8)(G/A/T/C)(C/T)GIACI(G/A)(T/C)CAT-3′ SJL197:5′-   CCGGAATTC(A/G)CAI(C/G)C(A/G)CAI (SEQ ID NO:9)(A/C)G(G/A/T/C)(T/A)CIACI(G/A)(T/C)CAT-3′ SJL198:5′-   CCGGAATTC(A/G)CAI(C/G)C(A/G)CAI(A/C)G (SEQ ID NO:10)(G/A/T/C)(C/G/T)TIACI(G/A)(T/C)CAT-3′

[0082] PCR using each of these primer combinations was carried out withcDNA prepared from 0.4 μg poly A-selected RNA; reactions were carriedout at 94° C. for 1 minute, 50° C. for 2 minutes, and 72° C. for 2minutes for 40 cycles.

[0083] PCR products of approximately 280 base pairs were gel purified,digested with EcoRI, gel purified again, and subcloned into theBluescript vector (Stratagene, San Diego, Calif.). Bacterial coloniescarrying individual subclones were picked into 96 well microtiterplates, and multiple replicas were prepared by plating the cells ontonitrocellulose The replicate filters were hybridized to probesrepresenting known members of the family, and DNA was prepared fromnon-hybridizing colonies for sequence analysis.

[0084] Among the colonies analyzed in this manner was one thatrepresented a novel sequence, which was designated GDF-12. This murinesequence was subsequently used to analyze expression patterns and toisolate a human cDNA clone (see below).

EXAMPLE 2 Expression of GDF-12

[0085] To determine the expression pattern of GDF-12, RNA samplesprepared from a variety of adult tissues were screened by Northernanalysis. RNA isolation and Northern analysis were carried out asdescribed previously (Lee, S.-J., Mol. Endocrinol., 4:1034, 1990) exceptthat hybridization was carried out in 5×SSPE, 10% dextran sulfate, 50%formamide, 1% SDS, 200 μ/ml salmon DNA, and 0.1% each of bovine serumalbumin, ficoll, and polyvinylpyrrolidone. Five micrograms of twice polyA-selected RNA prepared from each tissue were electrophoresed onformaldehyde gels, blotted, and probed with GDF-12. As shown in FIG. 1,the GDF-12 probe detected a single mRNA species approximately 2.8 and1.9 kb in length, in adult liver.

EXAMPLE 3 Isolation of cDNA Clones Encoding GDF-12

[0086] In order to isolate cDNA clones encoding GDF-11, a cDNA librarywas prepared in the lambda ZAP II vector (Stratagene) using RNA preparedfrom human adult liver. From 5 μg of twice poly A-selected RNA preparedfrom human spleen, a cDNA library consisting of 20 million recombinantphage was constructed according to the instructions provided byStratagene. A portion of this library was screened without amplificationusing the murine GDF-12 PCR product as a probe. Library screening andcharacterization of cDNA inserts were carried out as describedpreviously (Lee, Mol. Endocrinol., 4:1034, 1990L), except that the finalwash was carried out in 2×SSC.

[0087] Partial sequence analysis of the first isolated clone showed thatit contained the entire coding sequence of GDF-12. A portion of thenucleotide and predicted amino acid sequence of this clone is shown inFIG. 2 and SEQ ID NOs:11 and 12. The sequence begins with a putativeproteolytic cleavage site which is followed by a C-terminal region of114 amino acids. The active C-terminal fragment is predicted to have amolecular weight of approximately 12,500.

[0088] The entire nucleotide sequence of the longest human GDF-12 cDNAclone isolated is shown in FIG. 3 and SEQ ID NO:13. The 2419 base pairsequence contains a single long open reading frame beginning with amethionine codon at nucleotides 218-2201 and extending for 350 codons.The sequence contains an in-frame stop codon upstream of the putativeinitiating methionine. The predicted amino acid sequence (SEQ ID NO:14)contains a stretch of hydrophobic amino acids near the N-terminussuggestive of a signal sequence for secretion, one potential N-linkedglycosylation site at amino acids 232-236 (box). The C-terminal regionfollowing the putative processing site (shaded box) contains all of thehallmarks present in other TGF-β family members (see above).

[0089] The C-terminal region following the predicted cleavage sitecontains all the hallmarks present in other TGF-β family members. GDF-12contains most of the residues that are highly conserved in other familymembers, including the seven cysteine residues with their characteristicspacing. Like the TGF-β's, and the inhibin β's, GDF-12 also contains twoadditional cysteine residues. In the case of TGF-β2 , these additionalcysteine residues are known to form an intramolecular disulfide bond(Daopin, et al., Science, 257:369, 1992; Schlunegger and Grutter,Nature, 358:430, 1992). A tabulation of the amino acid sequencehomologies between GDF-12 and the other TGF-β family members is shown inFIG. 4. Numbers represent percent amino acid identities between eachpair calculated from the first conserved cysteine to the C-terminus.

[0090] Athough the invention has been described with reference to thepresently preferred embodiment, it should be understood that variousmodifications can be made without departing from the spirit of theinvention. Accordingly, the invention is limited only by the followingclaims.

1 14 1 34 DNA Artificial sequence PCR primer 1 ccggaattcg gntggmgnvatggrtnrtnt aycc 34 2 33 DNA Artificial sequence PCR primer 2 ccggaattcrcanscrcanc ynwcnacnry cat 33 3 33 DNA Artificial sequence PCR primer 3ccggaattcr canscrcant snygnacnry cat 33 4 33 DNA Artificial sequence PCRprimer 4 ccggaattcr canscrcant snwcnacnry cat 33 5 33 DNA Artificialsequence PCR primer 5 ccggaattcr canscrcant snbtnacnry cat 33 6 33 DNAArtificial sequence PCR primer 6 ccggaattcr canscrcang mnygnacnry cat 337 33 DNA Artificial sequence PCR primer 7 ccggaattcr canscrcangmnwcnacnry cat 33 8 33 DNA Artificial sequence PCR primer 8 ccggaattcrcanscrcanm gnygnacnry cat 33 9 33 DNA Artificial sequence PCR primer 9ccggaattcr canscrcanm gnwcnacnry cat 33 10 33 DNA Artificial sequencePCR primer 10 ccggaattcr canscrcanm gnbtnacnry cat 33 11 360 DNA Homosapiens CDS (1)..(357) 11 cgg gcc agg agg agg acc ccc acc tgt gag cctgcg acc ccc tta tgt 48 Arg Ala Arg Arg Arg Thr Pro Thr Cys Glu Pro AlaThr Pro Leu Cys 1 5 10 15 tgc agg cga gac cat tac gta gac ttc cag gaactg gga tgg cgg gac 96 Cys Arg Arg Asp His Tyr Val Asp Phe Gln Glu LeuGly Trp Arg Asp 20 25 30 tgg ata ctg cag ccc gag ggg tac cag ctg aat tactgc agt ggg cag 144 Trp Ile Leu Gln Pro Glu Gly Tyr Gln Leu Asn Tyr CysSer Gly Gln 35 40 45 tgc cct ccc cac ctg gct ggc agc cca ggc att gct gcctct ttc cat 192 Cys Pro Pro His Leu Ala Gly Ser Pro Gly Ile Ala Ala SerPhe His 50 55 60 tct gcc gtc ttc agc ctc ctc aaa gcc aac aat cct tgg cctgcc agt 240 Ser Ala Val Phe Ser Leu Leu Lys Ala Asn Asn Pro Trp Pro AlaSer 65 70 75 80 acc tcc tgt tgt gtc cct act gcc cga agg ccc ctc tct ctcctc tac 288 Thr Ser Cys Cys Val Pro Thr Ala Arg Arg Pro Leu Ser Leu LeuTyr 85 90 95 ctg gat cat aat ggc aat gtg gtc aag acg gat gtg cca gat atggtg 336 Leu Asp His Asn Gly Asn Val Val Lys Thr Asp Val Pro Asp Met Val100 105 110 gtg gag gcc tgt ggc tgc agc tag 360 Val Glu Ala Cys Gly CysSer 115 12 119 PRT Homo sapiens 12 Arg Ala Arg Arg Arg Thr Pro Thr CysGlu Pro Ala Thr Pro Leu Cys 1 5 10 15 Cys Arg Arg Asp His Tyr Val AspPhe Gln Glu Leu Gly Trp Arg Asp 20 25 30 Trp Ile Leu Gln Pro Glu Gly TyrGln Leu Asn Tyr Cys Ser Gly Gln 35 40 45 Cys Pro Pro His Leu Ala Gly SerPro Gly Ile Ala Ala Ser Phe His 50 55 60 Ser Ala Val Phe Ser Leu Leu LysAla Asn Asn Pro Trp Pro Ala Ser 65 70 75 80 Thr Ser Cys Cys Val Pro ThrAla Arg Arg Pro Leu Ser Leu Leu Tyr 85 90 95 Leu Asp His Asn Gly Asn ValVal Lys Thr Asp Val Pro Asp Met Val 100 105 110 Val Glu Ala Cys Gly CysSer 115 13 2419 DNA Homo sapiens CDS (218)..(1267) 13 gagctgtgagggtcaagcac agctatccat cagatgatct actttcagcc ttcctgagtc 60 ccagacaatagaagacaggt ggctgtaccc ttggccaagg gtaggtgtgg cagtggtgtc 120 tgctgtcactgtgccctcat tggcccccag caatcagact caacagacgg agcaactgcc 180 atccgaggctcctgaaccag ggccattcac caggagc atg cgg ctc cct gat gtc 235 Met Arg LeuPro Asp Val 1 5 cag ctc tgg ctg gtg ctg ctg tgg gca ctg gtg cga gca cagggg aca 283 Gln Leu Trp Leu Val Leu Leu Trp Ala Leu Val Arg Ala Gln GlyThr 10 15 20 ggg tct gtg tgt ccc tcc tgt ggg ggc tcc aaa ctg gca ccc caagca 331 Gly Ser Val Cys Pro Ser Cys Gly Gly Ser Lys Leu Ala Pro Gln Ala25 30 35 gaa cga gct ctg gtg ctg gag cta gcc aag cag caa atc ctg gat ggg379 Glu Arg Ala Leu Val Leu Glu Leu Ala Lys Gln Gln Ile Leu Asp Gly 4045 50 ttg cac ctg acc agt cgt ccc aga ata act cat cct cca ccc cag gca427 Leu His Leu Thr Ser Arg Pro Arg Ile Thr His Pro Pro Pro Gln Ala 5560 65 70 gcg ctg acc aga gcc ctc cgg aga cta cag cca ggg agt gtg gct cca475 Ala Leu Thr Arg Ala Leu Arg Arg Leu Gln Pro Gly Ser Val Ala Pro 7580 85 ggg aat ggg gag gag gtc atc agc ttt gct act gtc aca gac tcc act523 Gly Asn Gly Glu Glu Val Ile Ser Phe Ala Thr Val Thr Asp Ser Thr 9095 100 tca gcc tac agc tcc ctg ctc act ttt cac ctg tcc act cct cgg tcc571 Ser Ala Tyr Ser Ser Leu Leu Thr Phe His Leu Ser Thr Pro Arg Ser 105110 115 cac cac ctg tac cat gcc cgc ctg tgg ctg cac gtg ctc ccc acc ctt619 His His Leu Tyr His Ala Arg Leu Trp Leu His Val Leu Pro Thr Leu 120125 130 cct ggc act ctt tgc ttg agg atc ttc cga tgg gga cca agg agg agg667 Pro Gly Thr Leu Cys Leu Arg Ile Phe Arg Trp Gly Pro Arg Arg Arg 135140 145 150 cgc caa ggg tcc cgc act ctc ctg gct gag cac cac atc acc aacctg 715 Arg Gln Gly Ser Arg Thr Leu Leu Ala Glu His His Ile Thr Asn Leu155 160 165 ggc tgg cat acc tta act ctg ccc tct agt ggc ttg agg ggt gagaag 763 Gly Trp His Thr Leu Thr Leu Pro Ser Ser Gly Leu Arg Gly Glu Lys170 175 180 tct ggt gtc ctg aaa ctg caa cta gac tgc aga ccc cta gaa ggcaac 811 Ser Gly Val Leu Lys Leu Gln Leu Asp Cys Arg Pro Leu Glu Gly Asn185 190 195 agc aca gtt act gga caa ccg agg cgg ctc ttg gac aca gca ggacac 859 Ser Thr Val Thr Gly Gln Pro Arg Arg Leu Leu Asp Thr Ala Gly His200 205 210 cag cag ccc ttc cta gag ctt aag atc cga gcc aat gag cct ggagca 907 Gln Gln Pro Phe Leu Glu Leu Lys Ile Arg Ala Asn Glu Pro Gly Ala215 220 225 230 ggc cgg gcc agg agg agg acc ccc acc tgt gag cct gcg accccc tta 955 Gly Arg Ala Arg Arg Arg Thr Pro Thr Cys Glu Pro Ala Thr ProLeu 235 240 245 tgt tgc agg cga gac cat tac gta gac ttc cag gaa ctg ggatgg cgg 1003 Cys Cys Arg Arg Asp His Tyr Val Asp Phe Gln Glu Leu Gly TrpArg 250 255 260 gac tgg ata ctg cag ccc gag ggg tac cag ctg aat tac tgcagt ggg 1051 Asp Trp Ile Leu Gln Pro Glu Gly Tyr Gln Leu Asn Tyr Cys SerGly 265 270 275 cag tgc cct ccc cac ctg gct ggc agc cca ggc att gct gcctct ttc 1099 Gln Cys Pro Pro His Leu Ala Gly Ser Pro Gly Ile Ala Ala SerPhe 280 285 290 cat tct gcc gtc ttc agc ctc ctc aaa gcc aac aat cct tggcct gcc 1147 His Ser Ala Val Phe Ser Leu Leu Lys Ala Asn Asn Pro Trp ProAla 295 300 305 310 agt acc tcc tgt tgt gtc cct act gcc cga agg ccc ctctct ctc ctc 1195 Ser Thr Ser Cys Cys Val Pro Thr Ala Arg Arg Pro Leu SerLeu Leu 315 320 325 tac ctg gat cat aat ggc aat gtg gtc aag acg gat gtgcca gat atg 1243 Tyr Leu Asp His Asn Gly Asn Val Val Lys Thr Asp Val ProAsp Met 330 335 340 gtg gtg gag gcc tgt ggc tgc agc tagcaagaggacctggggct ttggagtgaa 1297 Val Val Glu Ala Cys Gly Cys Ser 345 350gagaccaaga tgaagtttcc caggcacagg gcatctgtga ctggaggcat cagattcctg 1357atccacaccc caacccaaca accacctggc aatatgactc acttgacccc tatgggaccc 1417aaatgggcac tttcttgtct gagactctgg cttattccag gttggctgat gtgttgggag 1477atgggtaaag cgtttcttct aaaggggtct acccagaaag catgatttcc tgccctaagt 1537cctgtgagaa gatgtcaggg actagggagg gagggaggga aggcagagaa aaattactta 1597gcctctccca agatgagaaa gtcctcaagt gaggggagga ggaagcagat agatggtcca 1657gcaggcttga agcagggtaa gcaggctggc ccagggtaag ggctgttgag gtaccttaag 1717ggaaggtcaa gagggagatg ggcaaggcgc tgagggagga tgcttagggg acccccagaa 1777acaggagtca ggaaaatgag gcactaagcc taagaagttc cctggttttt cccaggggac 1837aggacccact gggagacaag catttatact ttctttcttc ttttttattt ttttgagatc 1897gagtctcgct ctgtcaccag gctggagtgc agtgacacga tcttggctca ctgcaacctc 1957cgtctcctgg gttcaagtga ttcttctgcc tcagcctccc gagcagctgg gattacaggc 2017gcccactaat ttttgtattc ttagtagaaa cgaggtttca acatgttggc caggatggtc 2077tcaatctctt gacctcttga tccacccgac ttggcctccc gaagtgatga gattataggc 2137gtgagccacc gcgcctggct tatactttct taataaaaag gagaaagaaa atcaacaaat 2197gtgagtcata aagaagggtt agggtgatgg tccagagcaa cagttcttca agtgtactct 2257gtaggcttct gggaggtccc ttttcagggg tgtccacaaa gtcaaagcta ttttcataat 2317aatactaaca tgttatttgc cttttgaatt ctcattatct taaaattgta ttgtggagtt 2377ttccagaggc cgtgtgacat gtgattacat catctttctg ac 2419 14 350 PRT Homosapiens 14 Met Arg Leu Pro Asp Val Gln Leu Trp Leu Val Leu Leu Trp AlaLeu 1 5 10 15 Val Arg Ala Gln Gly Thr Gly Ser Val Cys Pro Ser Cys GlyGly Ser 20 25 30 Lys Leu Ala Pro Gln Ala Glu Arg Ala Leu Val Leu Glu LeuAla Lys 35 40 45 Gln Gln Ile Leu Asp Gly Leu His Leu Thr Ser Arg Pro ArgIle Thr 50 55 60 His Pro Pro Pro Gln Ala Ala Leu Thr Arg Ala Leu Arg ArgLeu Gln 65 70 75 80 Pro Gly Ser Val Ala Pro Gly Asn Gly Glu Glu Val IleSer Phe Ala 85 90 95 Thr Val Thr Asp Ser Thr Ser Ala Tyr Ser Ser Leu LeuThr Phe His 100 105 110 Leu Ser Thr Pro Arg Ser His His Leu Tyr His AlaArg Leu Trp Leu 115 120 125 His Val Leu Pro Thr Leu Pro Gly Thr Leu CysLeu Arg Ile Phe Arg 130 135 140 Trp Gly Pro Arg Arg Arg Arg Gln Gly SerArg Thr Leu Leu Ala Glu 145 150 155 160 His His Ile Thr Asn Leu Gly TrpHis Thr Leu Thr Leu Pro Ser Ser 165 170 175 Gly Leu Arg Gly Glu Lys SerGly Val Leu Lys Leu Gln Leu Asp Cys 180 185 190 Arg Pro Leu Glu Gly AsnSer Thr Val Thr Gly Gln Pro Arg Arg Leu 195 200 205 Leu Asp Thr Ala GlyHis Gln Gln Pro Phe Leu Glu Leu Lys Ile Arg 210 215 220 Ala Asn Glu ProGly Ala Gly Arg Ala Arg Arg Arg Thr Pro Thr Cys 225 230 235 240 Glu ProAla Thr Pro Leu Cys Cys Arg Arg Asp His Tyr Val Asp Phe 245 250 255 GlnGlu Leu Gly Trp Arg Asp Trp Ile Leu Gln Pro Glu Gly Tyr Gln 260 265 270Leu Asn Tyr Cys Ser Gly Gln Cys Pro Pro His Leu Ala Gly Ser Pro 275 280285 Gly Ile Ala Ala Ser Phe His Ser Ala Val Phe Ser Leu Leu Lys Ala 290295 300 Asn Asn Pro Trp Pro Ala Ser Thr Ser Cys Cys Val Pro Thr Ala Arg305 310 315 320 Arg Pro Leu Ser Leu Leu Tyr Leu Asp His Asn Gly Asn ValVal Lys 325 330 335 Thr Asp Val Pro Asp Met Val Val Glu Ala Cys Gly CysSer 340 345 350

1. Substantially pure growth differentiation factor-12 (GDF-12) andfunctional fragments thereof.
 2. An isolated polynucleotide sequenceencoding the GDF-12 polypeptide of claim
 1. 3. The polynucleotide ofclaim 2, wherein the GDF-12 is selected from the group consisting of: a.SEQ ID NO:13, wherein T can also be U; b. nucleic acid sequencescomplementary to SEQ ID NO:13; and c. fragments of a or b that are atleast 15 bases in length and that will selectively hybridize to DNAwhich encodes the GDF-12 protein of SEQ ID NO:14; and
 4. Thepolynucleotide of claim 2, wherein the polynucleotide is isolated from amammalian cell.
 5. The polynucleotide of claim 4, wherein the mammaliancell is selected from the group consisting of mouse, rat, and humancell.
 6. An expression vector including the polynucleotide of claim 2.7. The vector of claim 6, wherein the vector is a plasmid.
 8. The vectorof claim 6, wherein the vector is a virus.
 9. A host cell stablytransformed with the vector of claim
 6. 10. The host cell of claim 9,wherein the cell is prokaryotic.
 11. The host cell of claim 9, whereinthe cell is eukaryotic.
 12. Antibodies that bind to the polypeptide ofclaim 1 or fragments thereof.
 13. The antibodies of claim 12, whereinthe antibodies are polyclonal.
 14. The antibodies of claim 12, whereinthe antibodies are monoclonal.
 15. A method of detecting a cellproliferative disorder comprising contacting the antibody of claim 12with a specimen of a subject suspected of having a GDF-12 associateddisorder and detecting binding of the antibody.
 16. The method of claim15, wherein the cell is a liver cell.
 17. The method of claim 15,wherein the detecting is in vivo.
 18. The method of claim 17, whereinthe antibody is detectably labeled.
 19. The method of claim 18, whereinthe detectable label is selected from the group consisting of aradioisotope, a fluorescent compound, a bioluminescent compound and achemiluminescent compound.
 20. The method of claim 15, wherein thedetection is in vitro.
 21. The method of claim 20, wherein the antibodyis detectably labeled.
 22. The method of claim 21, wherein the label isselected from the group consisting of a radioisotope, a fluorescentcompound, a bioluminescent compound, a chemoluminescent compound and anenzyme.
 23. A method of treating a cell proliferative disorderassociated with expression of GDF-12, comprising contacting the cellswith a reagent which suppresses the GDF-12 activity.
 24. The method ofclaim 23, wherein the reagent is an anti-GDF-12 antibody.
 25. The methodof claim 23, wherein the reagent is a GDF-12 antisense sequence.
 26. Themethod of claim 23, wherein the cell is a liver cell.
 27. The method ofclaim 23, wherein the reagent which suppresses GDF-12 activity isintroduced to a cell using a vector.
 28. The method of claim 27, whereinthe vector is a colloidal dispersion system.
 29. The method of claim 28,wherein the colloidal dispersion system is a liposome.
 30. The method ofclaim 29, wherein the liposome is essentially target specific.
 31. Themethod of claim 30, wherein the liposome is anatomically targeted. 32.The method of claim 31, wherein the liposome is mechanisticallytargeted.
 33. The method of claim 32, wherein the mechanistic targetingis passive.
 34. The method of claim 32, wherein the mechanistictargeting is active.
 35. The method of claim 34, wherein the liposome isactively targeted by coupling with a moiety selected from the groupconsisting of a sugar, a glycolipid, and a protein.
 36. The method ofclaim 35, wherein the protein moiety is an antibody.
 37. The method ofclaim 36, wherein the vector is a virus.
 38. The method of claim 37,wherein the virus is an RNA virus.
 39. The method of claim 38, whereinthe RNA virus is a retrovirus.
 40. The method of claim 39, wherein theretrovirus is essentially target specific.
 41. The method of claim 40,wherein a moiety for target specificity is encoded by a polynucleotideinserted into the retroviral genome.
 42. The method of claim 40, whereina moiety for target specificity is selected from the group consisting ofa sugar, a glycolipid, and a protein.
 43. The method of claim 42,wherein the protein is an antibody.